The ARH1 knockout mouse and cells grown from knockout animals were further characterized to assess the potential importance of ADP-ribose-(arginine)-proteins in cellular regulation. Cells lacking the ARH1 gene proliferated faster than did ARH1-/- cells transformed with a wild-type ARH1 gene, whereas cells over-expressing a mutant ARH1 (D60A/D61A), which encodes a protein with < 0.1 % of wild-type activity, showed enhanced proliferation similar to ARH1-/- cells. ARH1-/- cells also produced colonies in soft agar, as did ARH1-/- cells transformed with the ARH1 gene containing the double mutation. In contrast, ARH1-/- cells transformed with the wild-type gene did not form colonies in soft agar. Further consistent with the tumorigenic potential of ARH1-/- cells, those and the cells transformed with the mutated ARH1 gene also formed tumors in nude mice, whereas ARH1-/- cells transformed with the wild-type gene did not. Of importance, ARH1+/- cells from heterozygous animals generated tumors in nude mice. The ARH1 gene from those tumors was sequenced, which revealed mutations. Thus, the ARH1 gene appeared to determine, in part, the tumorigenic potential of those cells. We also looked at the ARH1 knockout mouse for additional information regarding the potential of the ARH1 gene to regulate cell proliferation and tumorigenesis. Wild-type, heterozygous, and ARH1-/- knockout mice differed in the frequency and types of tumors formed. Both heterozygous and ARH1-/- knockout mice developed tumors, including lymphosarcoma, rhabdomyosarcoma, and adenocarcinoma in excess of wild-type mice and at an earlier age. In some instances, metastases were identified. To follow tumor development in the animals, magnetic resonance imaging of the abdomen and thorax was used. In the heterozygous mice, the single ARH1 gene from the tumors was sequenced to reveal a potential mutation, not found in the wild-type animals or in non-tumor tissue from the heterozygous mouse. These data suggest that the ADP-ribosylation of arginine residues on protein may be critical in cellular homeostasis and that the lack of the ARH1 gene may accelerate tumor formation.